Project 1: An RNAi screen of microtubule-regulatory proteins identifies MARK2/Par1 as an effector of Rac1-mediated microtubule growth. Yukako Nishimura, Kathryn Applegate, Gaudenz Danuser, Clare Waterman Proper regulation of microtubule (MT) assembly dynamics is essential for directed cell migration. Microtubule dynamics in migrating cells are spatially regulated by Rho GTPases. We have previously shown that activated Rac1 induces MT net growth by suppressing catastrophe and increasing growth velocity, and that Rac1 activity is required for polarized MT growth in the leading edge of migrating cells. We identified a necessary, but not sufficient PAK kinase-mediated pathway downstream of Rac1 that promoted MT growth. Therefore, we hypothesized that additional factors promote MT net growth downstream of Rac1. To find these factors, we performed a RNAi screen in human U2OS osteosarcoma cells to determine if known MT-regulatory proteins were required for constitutively activated Rac1 promotion of MT growth. To analyze MT dynamics, we imaged fluorescent-tagged EB3, a MT plus-end binding protein that serves as a probe for the position of MT ends, and tracked the motion of EB3 comets in time-lapse movies using an automated computer program. Our results indicate that depletions of several MT-binding proteins change the growth rate of MT in activated Rac1-expressing cells. We have focused on MARK2, a microtubule affinity-regulating kinase homologous to the C. elegans polarity protein Par1, whose depletion reduces the number of elongated MTs in the leading edge of Rac1-activated cells. We are currently testing how MARK2 is involved in promoting MT growth downstream of Rac1 and it requirement in cell migration. Project 2: MCAK Activity Controls Interphase Microtubule Dynamics and Directed Cell Migration. Myers, K.A.;Applegate, K;Danuser G.;and Waterman, C.M. Directional cell migration is initiated through extracellular stimuli that coordinate changes in the cytoskeleton to establish a polarized cellular morphology. Cell polarity can be achieved through regional regulation of microtubule (MT) dynamics, including MT growth toward the leading edge and MT shortening in the cell rear. Mitotic Centromere Associated Kinesin (MCAK) is a MT depolymerase that is down-regulated in mitosis by Aurora kinase phosphorylation. While its mitotic functions have been well-characterized, whether MCAK regulates MT dynamics during cell migration is not known. We hypothesize that MCAK is down-regulated locally via a Rac1/Pak1/Aurora-A kinase signaling pathway to establish preferential MT growth toward the leading edge and to promote MT shortening within the cell rear. To test this hypothesis, we performed time-lapse imaging of fluorescently tagged EB3 as a marker of MT plus end growth in HUVEC cells and analyzed MT dynamics and cell behavior under different manipulations of the proposed signaling cascade. We find that MCAK knockdown (KD) produces expected effects on the MT cytoskeleton, including increased levels of tubulin polymer and decreased MT catastrophe frequency. MCAK-KD cells show a reduction in MT polymerization speeds and exhibit a mal-oriented MT array, as well as a statistically significant reduction in cell migration velocity, directional persistence, and distance to origin, indicating a defect in cell migration and/or polarization. These effects are rescued through expression of exogenous wild-type-MCAK, but not by expression of either an inactive (ATPase-dead) MCAK mutant or an MCAK mutant that is incapable of phospho-regulation by Aurora-A kinase. Immunolabeling of cells expressing either constitutively active-Rac1 or constitutively active-Pak1 suggests that Rac1 and Pak1 activities correlate with increased Aurora-A activity, as assayed with a phospho-specific antibody, and also correlate with decreased levels of MCAK expression. These data suggest that interphase regulation of MCAK is achieved downstream of a Rac1/Pak1/Aurora-A signaling pathway in order to locally coordinate MCAK-mediated MT depolymerization as a method to ensure proper cell polarization and motility. Project 3: Regulation of microtubules in migrating endothelial cells in 3D ECMs Ken Myers, Kathryn Applegate, Gaudenz Danuser, Clare Waterman ECM dimensionality and stiffness regulate endothelial cell branching morphogenesis via mechanosensitive cell adhesion receptors that elicit bidirectional signals to and from the actomyosin cytoskeleton. Stiff ECMs promote myosin II activity that inhibits cell branching, while compliant ECMs reduce myosin II activity and promote branching. The MT cytoskeleton controls cell morphology through its structural and regulatory interactions with actomyosin. However the role of MTs in cellular responses to ECM properties, and the effects of ECM properties on MT organization and dynamics are unknown. To explore the role of MTs in cell responses to ECM dimensionality and compliance, we analyzed MT and actin organization in HUVEC cells on two different stiffnesses of 2D and 3D ECMs. This showed that stiffer substrates enhanced MT presence in cell branches. Depolymerization of MTs with nocodazole induced actin stress fibers and inhibited cell branching independent of substrate stiffness or dimensionality, showing that MT depolymerization-induced activation of contractility is not regulated by physical properties of ECM. In contrast, stabilization of MTs with taxol inhibited MT presence in branches. To determine how ECM stiffness and dimensionality affect MTs, we analyzed MT dynamics by tracking fluorescently-tagged MT plus ends. This revealed that on soft substrates, MT growth rate was independent of ECM dimensionality, while on stiff substrates, MT growth was faster in 3D compared to 2D ECMs. In 2D, MT growth rate was independent of stiffness, while in 3D, stiffer ECM promoted faster MT growth than soft ECM. Thus, the fastest MT growth occurred in stiff 3D ECMs, and the slowest occurred in 2D ECMs, independent of stiffness. Inhibition of myosin II with blebbistatin increased MT growth rate under all conditions of ECM dimensionality and stiffness, indicating that myosin II regulates MT growth independent of ECM physical properties. Furthermore, blebbistatin did not abrogate the effects of ECM stiffness on MT growth. Thus, we find a bidirectional regulation scheme where MTs mediate cellular responses to ECM, and ECM regulates MTs by both contractility-dependent and independent mechanisms.